Pipetting Techniques and Gel Electrophoresis - Study Notes

Pipetting Techniques

  • Types of micropipettes

    • Air displacement pipette (most commonly used)
    • An air cushion is located between the piston and the liquid.
    • When the push button is pressed, the piston moves to expel a volume of air.
    • The volume of air displaced is equivalent to the volume of liquid aspirated; i.e., V<em>extair=V</em>extliquid.V<em>{ ext{air}} = V</em>{ ext{liquid}}.
    • Positive displacement pipette
    • Piston is in contact with the liquid; works like a syringe.
    • The aspiration force remains constant and is unaffected by the physical properties of the sample.
    • The piston is disposable.

  • Parts of a pipette and pipette tip

    • Piston
    • Piston spring
    • Compression set
    • Compression bush
    • Seal and O-ring
    • Ejector
    • Identification mark
    • Pushbutton
    • Volume adjustment knob + locking ring
    • Shaft nut
    • Tip ejector
    • Shaft
    • Filter level (if present)
    • Mark (identification)
    • Pushbutton + knob (usage for volume control)
    • Sealing ring and O-ring

  • Use of Air Displacement Pipettes

    1. Set the volume to the required value.
    • The piston moves to the appropriate position.
    1. Prepare for aspiration.
    • Press the push-button prior to sample aspiration; the piston descends and expels a volume of air equal to the selected volume of liquid.
    1. Aspirate the sample.
    • As the push-button is released, a partial vacuum is created inside the tip; ambient atmospheric pressure pushes the desired volume of liquid through the orifice into the tip.
    1. Dispense the sample.
    • Press the push-button again; air pressure increases inside the shaft and tip, pushing the liquid out of the tip.

  • Use of Positive Displacement Pipettes

    1. Set the volume.
    • The required volume is set; the piston moves down to the appropriate start position.
    1. Prepare for aspiration.
    • Press the push-button prior to sample aspiration; the piston descends to the end of the capillary.
    1. Aspirate the sample.
    • The orifice is immersed below the liquid surface; as the push-button is released, the piston moves up and ambient pressure forces the liquid through the orifice into the capillary.
    1. Dispense the sample.
    • Press the push-button again; the piston moves down and expels the liquid out of the capillary.

  • Modes of Pipetting

    • Forward pipetting
    • Preferred when dispensing aqueous solutions (low detergents and low protein content).
    • Reverse pipetting
    • Usually performed when dispensing viscous or foaming liquids, or when dispensing very small volumes.

  • Forward Pipetting (step-by-step)

    1. Preparation: Hold the instrument in a nearly vertical position; depress the plunger smoothly to the first stop position.
    2. Aspiration: Immerse the pipette tip in the liquid; allow the plunger to move up smoothly to the rest position; wait one second for liquid to be drawn into the tip.
    3. Distribution: Place the tip at an angle of about 10exto10^ ext{o}–45exto45^ ext{o} against the inside wall of the receiving vessel; depress the plunger smoothly to the first stop.
    4. Purge (blow-out): Wait one second, then depress the plunger to the second stop to remove any remaining sample from the tip. Remove the tip from the sidewall by sliding it up.
    5. Home: Allow the plunger to move back to the rest position.
    • Ready position, First stop, Second stop (purge) indicated on the diagram for reference.

  • Reverse Pipetting (step-by-step)

    1. Preparation: Hold the instrument in a nearly vertical position; depress the plunger smoothly to the second stop position.
    2. Aspiration: Immerse the tip in the liquid; allow the plunger to move up smoothly to the rest position; wait one second for liquid to fill.
    3. Distribution: Place the tip at an angle (≈10exto10^ ext{o}–45exto45^ ext{o}) against the inside wall of the receiving vessel; depress the plunger smoothly to the first stop; wait one second.
    4. Re-aspiration: If reusing the tip for the same sample, keep the plunger in the intermediate position for the next immersion and restart operation 2.
    5. Complete purge: Wait one second and purge. If the tip will not be reused, depress the plunger to the purge position over an appropriate waste container and eject the tip.
    • Rest position, First stop, Second stop (purge) shown in the diagram.

  • Recommended Pipette Types and Techniques

    • Solution/compound: Aqueous solution (buffers, diluted salt solutions)
    • Pipette: Air displacement
    • Tip: Standard
    • Technique: Forward
    • Solution/compound: Viscous solution (protein and nucleic acid solutions, glycerol, Tween 20/40/60/8)
    • Pipette: Positive displacement
    • Tip: Standard or wide orifice
    • Technique: Reverse
    • Solution/compound: Volatile compounds (e.g., methanol, hexane)
    • Pipette: Air displacement
    • Tip: Filter
    • Technique: Forward
    • Solution/compound: Nucleotide solutions (genomic DNA, PCR products)
    • Pipette: Air displacement or Positive displacement
    • Tip: Filter or wide orifice
    • Technique: Forward
    • Solution/compound: Radioactive compounds (carbonate, 3H-thymidine)
    • Pipette: Air displacement or Positive displacement
    • Tip: Filter
    • Technique: Forward
    • Solution/compound: Acids/alkalis (H2SO4, HCl, NaOH)
    • Pipette: Air displacement or Positive displacement
    • Tip: Filter
    • Technique: Forward
    • Solution/compound: Toxic samples
    • Pipette: Air displacement or Positive displacement
    • Tip: Filter
    • Technique: Forward or reverse
    • For genomic DNA, wide-orifice tips can be used to eliminate mechanical shearing
    • Notes: Always consider bubble formation; pipette slowly for samples prone to shear, and use vapour barriers/filters when handling volatile or toxic compounds.

  • Pipetting Technique Considerations

    • Pre-rinsing or pre-wetting the pipette tip provides greater uniformity and precision of dispensing.
    • Aspirate the liquid to be dispensed with the tip, then dispense back to the original reservoir or to waste.
    • Perform 2 to 3 times.
    • Perform every time the tip is changed or if the volume setting is increased.

  • Pipetting Technique Considerations (Blood Sample Example)

    • In vertical position, press the plunger up to the first stop.
    • Immerse the pipette tip into the whole blood sample.
    • Release the plunger slowly to aspirate the fluid.
    • Into the receiving chamber, press the plunger slowly up to the first stop to dispense the liquid. Repeat until the interior of the tip is clear.
    • Press the plunger up to the second stop to completely empty the tip.
    • Remove the pipette from the receiving container and slowly release the plunger.
    • Press the eject button to dispose of the tip.

  • General Pipetting Considerations

    1. Organize things and ensure all reagents are present prior to working.
    2. Sterilize the working area and materials to be used.
    3. Choose the right pipette.
    4. Make sure the tip is properly mounted and fitted before the volume is set.
    5. Adjust the volume before pipetting.
    6. Choose mode of pipetting based on the type of sample.
    7. Pipetting is always done with the pipette held in a vertical position.
    8. Pipetting is done slowly with a continuous pace.
    9. Once done, eject used tip into the correct trash bin.
    10. Store the pipette in an upright position.

  • Pipetting Resources

    • Online simulation of pipetting: https://www.labxchange.org/library/pathway/lx-pathway:cbb4baba-67d9-41d2-8518-6abc4ad96ca0/items/lb:LabXchange:4eecf5fe:lx_simulation:

  • Electrophoresis: Overview

    • Separation of DNA, RNA, or protein molecules via electric current.
    • An electric current is applied to promote movement of molecules through a matrix.
    • The sample or gel is stained and visualized; nucleic acids are stained using fluorescent visualization dyes and viewed via a gel documentation viewer.
    • Molecules are separated by size and charge: smaller molecules travel farther; positively charged molecules move toward the cathode; negatively charged molecules move toward the anode.

  • Nucleic acids and electrophoresis specifics

    • DNA and RNA are negatively charged and travel toward the anode when electrophoresed.
    • Nucleic acid size is expressed in base pairs (bp).
    • Unknown bp samples are compared with a ladder containing at least ten (10) known bp values.

  • Components of an electrophoresis system

    • Power supply – provides the electric current.
    • Buffer tank – holds the gel and the running buffer.
    • Cathode – negative electrode (current source).
    • Anode – positive electrode (current travels toward).

  • Gels and reagents

    • Gel – matrix through which samples migrate; types:
    • Agarose gel – used for most nucleic acid electrophoresis applications.
    • Polyacrylamide gel (PAGE) – used for protein electrophoresis and sometimes for small DNA fragments.
    • Running buffer – conducts current and maintains pH.
    • Common buffers:
    • Tris-acetate-EDTA (TAE): used to separate DNA fragments ext{> } 2000 ext{ bp}.
    • Tris-borate-EDTA (TBE): used to separate DNA fragments ext{< } 2000 ext{ bp}. TBE is more conductive and suitable for longer runs. Borate inhibits enzymatic activity.
    • Nucleic acid ladders – solutions with DNA/RNA of known bp lengths.
    • Loading dye – enables visual tracking of samples through the gel; dyes include extBromophenolblue,extXylenecyanolFF,extOrangeGext{Bromophenol blue}, ext{Xylene cyanol FF}, ext{Orange G}.
    • Visualization dyes – fluorescent dyes used to detect DNA in a gel documentation viewer; examples include extEthidiumbromide,extSYBRGreen,extGelRedext{Ethidium bromide}, ext{SYBR Green}, ext{Gel Red}.

  • Procedure for DNA Electrophoresis (example workflow)

    1. Prepare 1000 extmL1000 \, ext{mL} of 1X TAE buffer: add 20 extmL20 \, ext{mL} of 50X TAE buffer to 980 extmL980 \, ext{mL} of molecular-grade water.
    2. Prepare 100 mL of 1% agarose gel by weighing 1 g of agarose powder and adding to 100 mL of 1X TAE buffer in an Erlenmeyer.
    3. Heat the mixture in a microwave until dissolved; check every 30 seconds until clear; do not allow boiling.
    4. While gel is liquid, add 10 extµL10 \, ext{µL} of Gel Red; swirl to mix.
    5. Pour the gel into the casting mold and allow to set.
    6. Once set, transfer the gel into the electrophoresis chamber/buffer tank with wells near the cathode.
    7. Pour 1X extTAE1X \, ext{TAE} buffer into the tank up to the mark; ensure the gel is fully submerged.
    8. Prepare loading samples by mixing 5 extµL5 \, ext{µL} of DNA sample with 1 extµL1 \, ext{µL} of loading dye.
    9. Pipette 5 extµL5 \, ext{µL} of ladder and loading samples into the assigned wells.
    10. Cover the chamber and connect to the power supply.
    11. Run the gel at 90extV90 ext{ V} for 60extmin60 ext{ min}.
    12. When finished, disconnect and remove the chamber lid.
    13. Transfer the gel to a gel visualization tray.
    14. Visualize the gel under UV light in a gel documentation viewer.

  • Electrophoresis online simulation

    • Online resource: LabXchange simulation link (same as above) for virtual practice.

  • Practical notes and concepts

    • Smaller fragments migrate faster through the gel matrix than larger fragments due to size-based sieving.
    • Charge polarity governs direction of migration: nucleic acids are negatively charged and migrate toward the anode.
    • Gel composition (agarose vs polyacrylamide) affects resolution; choose based on fragment size and molecule type.
    • Visualizing dyes must be compatible with the detection method; some dyes are non-mutagenic (e.g., Gel Red) compared to ethidium bromide.

  • Ethical and safety considerations (implicit in lab practice)

    • Proper handling of potentially hazardous reagents (e.g., Ethidium bromide and toxic dyes) and radioactive substances.
    • Use of appropriate personal protective equipment and waste disposal per institutional guidelines.
    • Accurate labeling and record-keeping for ladders and samples to avoid cross-contamination.

  • Connections to foundational principles

    • The dependence of electrophoretic mobility on size and charge reflects fundamental physics of polymer gels and electrostatics.
    • Pipetting accuracy and precision directly impact experimental reproducibility and data interpretation in molecular diagnostics.
    • Understanding buffer systems (TAE vs TBE) links to buffering capacity, ionic strength, and effects on DNA migration and enzyme activity.

  • Notable formulas and constants

    • Relationship between aspirated liquid and displaced air in air-displacement pipettes: V<em>extair=V</em>extliquid.V<em>{ ext{air}} = V</em>{ ext{liquid}}.
    • Common volumes and steps presented in the protocol are given as exact quantities such as:
    • 1000 mL1000 \, \mathrm{mL} of 1X TAE buffer; 20 mL20 \, \mathrm{mL} of 50X TAE buffer; 980 mL980 \, \mathrm{mL} water.
    • Gel composition: 1%1\% agarose in 100 mL100 \, \mathrm{mL} 1X TAE buffer.
    • Loading: 5μL5 \mu L DNA sample with 1μL1 \mu L loading dye; ladder: 5μL5 \mu L.
    • Run condition: 90 V90 \, \mathrm{V} for 60min60 \mathrm{min}.

  • Quick reference cheat sheet

    • Pipette types: Air displacement (most general) vs Positive displacement (viscous/volatile samples).
    • Pipetting modes: Forward (aqueous) vs Reverse (viscous/foamy).
    • Typical preparation steps for gel electrophoresis: buffer prep, gel casting, loading, running, visualization.

  • Practical tips for exams

    • Memorize the typical sequence for forward and reverse pipetting; know the meaning of first stop, second stop (purge).
    • Be comfortable with common buffer compositions and their uses: TAE vs TBE; know that borate can inhibit enzymatic activity.
    • Remember typical run conditions and common safety steps (tip ejection and waste disposal).

  • Online resources

    • Gel electrophoresis and pipetting simulations available through LabXchange and related educational platforms (see links above in the Pipetting Resources section).